PROJECT SUMMARY Current antiretroviral therapy effectively suppresses but does not eradicate HIV-1 infection. During combination antiretroviral therapy (cART), reduction of HIV-1 RNA levels to less than 50 copies/ml is frequently achieved; however, persistent low-level viremia has been detected in plasma using ultrasensitive assays. The source and dynamics of this persistent viremia is currently under investigation. One well-defined reservoir of HIV-1 is memory CD4+ T cells, where HIV-1 latency is established when an activated CD4+ T cell becomes infected by HIV-1 but transitions to a terminally differentiated memory T cell instead of undergoing lytic infection. Upregulating cellular transcription to induce HIV gene expression has been proposed as a strategy for reducing the pool of latently- infected resting memory CD4+ T cells and possibly myeloid cells carrying an integrated form of the viral genome. This includes inhibiting cellular histone deacetylases (HDACs) because these HDACs promote latency by regulating genome structure and transcriptional activity. A recent treatment of eight HIV-infected patients on suppressive cART with a single dose of the histone deacetylase inhibitor (HDACi), vorinostat, resulted in a significant increase in cell associated unspliced (CA-US) HIV RNA in resting memory CD4+ T cells. During a clinical trial in Australia, a similar significant increase in CA- US HIV RNA was observed in CD4+ T-cells in blood from HIV-infected patients treated with 14 days of the HDACi vorinostat. Preliminary data from a multidose study of the HDACi panobinostat in Denmark also demonstrates an increase in CA-US HIV RNA. Given these findings, we hypothesize that: 1) the sequence of CA-US HIV RNA following HDACi treatment will be clonal which is consistent with transcription from a subset of infected resting memory T cells responsive to HDAC inhibitors in the blood and gut; 2) specific cell types are producing CA-US HIV RNA; 3) this CA-US HIV RNA will be genetically similar to pre-therapy (pre-cART and pre-HDACi) plasma-derived HIV-1 sequences, indicating this reservoir was established prior to cART initiation; 4) if virus rebounds during an analytical treatment interruption post-panobinostat therapy, it will be genetically similar to CA-US HIV RNA reactivated during HDACi therapy. These hypotheses will be addressed in the experiments of the following Specific Aims: (1) to determine the genetic makeup and diversity of CA-US HIV RNA from CD4+ T cells of patients prior to and following multi- dose treatment with vorinostat or panobinostat; (2) to determine the genetic makeup and diversity of unspliced HIV-1 RNA in myeloid cells and specific memory CD4+ T cell subsets; and (3) to compare the genetic makeup of CA-US HIV RNA to plasma-derived HIV-1 sequences from pre-therapy samples and samples taken after an analytical treatment interruption following panobinostat therapy. If this exploratory study reveals clonal expansion of CA-US HIV RNA in myeloid cells and specific resting memory T cells from HIV-1 infected patients on HDAC inhibitors, this would provide direct evidence that these compounds are disrupting proviral latency within these cells and that HIV latency can be therapeutically targeted in humans.